Method for isolating, in particular for detecting or quantifying an analyte in a medium

ABSTRACT

The present invention relates to a method for at least one of detecting, quantifying or isolating at least one analyte from a liquid medium in which the analyte is distributed and comprises providing a reagent comprised of particles having a receptor for an analyte fixed to the particles distributed in the medium and a capturing means having an exposed surface defining an active zone. An intermediate reagent is formed by the complex of the reagent with the analyte. A second receptor is fixed in the active zone to capture either the analyte bound by the reagent or the receptor (capture partners). The active zone serves as a site of isolation and concentration of the capture partners.

BACKGROUND

The present invention relates to a method for isolating at least oneanalyte from a liquid medium, in which the latter is distributed.

“Isolating” or “isolation” is generically understood to mean anytechnique which makes it possible to separate said analyte, but also toenrich or concentrate, in relation to said analyte, any liquid mixturecontaining it. However, it is also understood to mean, optionallyconjointly with the preceding definition, any technique which makes itpossible to determine the analyte, for the purpose of a detection and/orquantification thereof, in the liquid medium containing it.

“Analyte” is understood to mean any entity, in particular biologicalentity, to be isolated. Among the range of the analytes considered belowby the present invention, there may be mentioned cells, organelles,viruses and bacteria, antibodies, antibody fragments, antigens, haptens,lectins, sugars, ribodeoxyribo-nucleic acids, proteins, in particular Aor G, hormones, hormone receptors, biotin, avidin, streptavidin and ingeneral all natural or synthetic molecules or macromolecules, oranalogs, to be determined, that is to say detected and/or quantified.

In accordance with the document FR-A-2,679,660, a method is known forisolating at least one analyte, namely either an antigen which is freeor bound to the surface of a cell, or an antibody directed against acellular or tissue antigen, for example anti-erythrocyte antibody.

According to this method, at least one reagent is available whichcomprises, on the one hand, a free support, in discrete form, in thiscase relatively large-sized magnetic particles consisting of a polymerwhich encapsulates ferrite granules, and, on the other hand, exhibitinga receptor for the analyte, namely either an antibody, or an antigen orrevealing substance, for example an anti-human immunoglobulin antibody.

Another support, for capture, is also available which consists of thewall of a container or well, whose accessible surface comprises at leastone useful or active zone, of limited surface, exhibiting at least oneother receptor for an entity to be captured, namely for said analyte orfor said receptor. This other receptor is either an antibody, or anantigen, for example of the erythrocyte type.

According to this method, the accessible surface of said other supportis brought into contact with the analyte, contained in a liquid sample,in order to bind the analyte to the accessible surface, and to assembleit in the form of a deposit on said surface, immobilized on said othersupport via said other receptor.

Next, for revealing purposes, the reagent is brought into contact withthe immobilized analyte, in order to obtain with the latter acombination, also immobilized on said other support and capable ofallowing the isolation or determination of the analyte.

A method as defined above is therefore carried out with a reagentconsisting of a support in discrete form, to which at least one ligandforming a receptor for the analyte is bound.

Preferably, these particles are magnetic. They can be classified intotwo categories, namely particles of relatively large diameter, forexample of the order of one or a few micron(s), and those of relativelysmall diameter, for example of the order of a few tens of nanometers,and in the colloidal state.

The magnetic particles of relatively large diameter, when they areplaced in a magnetic field, move in the direction of the site where thefield is highest and at a sufficient speed to be separated from theirmedium by this means.

By way of example, there may be mentioned the particles described in thedocument EP-A-0,125,995. They are obtained by precipitation of ferrousand ferric salts in basic medium, followed by a silanization reaction inmethanol. Their final diameter is between 0.1 and 1.5 μm and theirdensity is 2.5 g/cm³. Likewise, the particles described in the documentsEP-A-0,106,873, EP-A-0,585,868 and U.S. Pat. No. 5,356,713 are obtainedby various methods of polymerization, or alternatively those describedin the document U.S. Pat. No. 4,297,337 use a porous glass matrix inwhich magnetic pigments are dispersed. Other patents also describe theuse of small-sized particles, but deliberately aggregated in order toincrease the magnetic mass as in the document U.S. Pat. No. 5,169,754.The article by P. A. Risφen et al, Protein Expression and Purification,6 (1995), 272-277 also describes magnetic gels.

Placed in a magnetic field, all these relatively large particlesgenerate a movement in the direction of the side where the field is themost intense. A simple permanent magnet or equivalent assemblies asdescribed for example in the document EP-A-0,317,286 or U.S. Pat. No.565,665 may be used. These particles are commonly used for theseparation of cells or of molecules, as well as in immunoassays asdescribed in the document EP-A-0,528,708 but are not used as systems fordetection and quantification.

Moreover, their diameter and their density are such that they sedimentvery rapidly under the effect of gravity, which makes them difficult touse because of the stirring stresses or lack of homogeneity of thesolutions due to the creation of a concentration gradient.

In summary, the relatively large particles are difficult to use and arenot very appropriate for the determination of an analyte.

Magnetic particles of relatively small diameter are practically notattracted by a simple permanent magnet within reasonable periods: theseparticles are in particular widely used for the magnetic separation ofcells. These particles are known to persons skilled in the art by thename of “superparamagnetic” particles. “Superparamagnetic” particles areunderstood to mean particles whose diameter is too small to consist ofseveral magnetic domains. They are characterized by a high magneticsusceptibility and a high magnetization at saturation, but a zero orvery low magnetic stability. These particles are in particular widelyused for the magnetic separation of cells. For example, those describedin the document U.S. Pat. No. 4,230,685 are obtained by emulsifying amixture of albumin, of protein A and of particles of Fe₃O₄ 15-20 nm indiameter and can immobilize antibodies via the protein A. The documentU.S. Pat. No. 4,452,773 describes another type of particle, obtained byprecipitation of ferrous and ferric salts in a basic medium, and in thepresence of a polysaccharide. These particles can immobilize antibodies,oligonucleotides, lectins or other biomolecules, by coupling to thepolysaccharide by means of known methods of grafting. Their use hasoften been repeated, as in the documents U.S. Pat. No. 5,543,289 orWO-A-88/00060, or they are used in specific applications such as thosedescribed in the documents FR-A-2,710,410 and FR-A-2,732,116. Thedocument U.S. Pat. No. 4,795,698 describes a modification of the Moldayprocedure, by replacing, for example, the polysaccharide with anotherpolymer of a protein nature. The proteins present at the surface of theparticles can thus serve for the subsequent immobilization of antibodiesby coupling methods known to persons skilled in the art.

These particles require the use of special assemblies which make itpossible to locally increase the magnetic field gradient. This techniqueis known by persons skilled in the art by the name of HGMS (for HighGradient Magnetic Separation) and it is for example described by WO96/09409. It uses a device discrete form and for this reason are notused for the separation, concentration or enrichment of an analyte.

Furthermore, after separation, the particles are rather considered as aninconvenience in the subsequent steps of any method. In the documentU.S. Pat. No. 4,018,886, they are even removed deliberately. Thedocument FR-A-2,710,410 uses the presence of small-sizedsuperparamagnetic particles as detection components which make itpossible to quantify a molecular recognition reaction. In this case, theprinciple used is an agglutination reaction resulting in the formationof an aggregate. Furthermore, the articles are not used as a means ofconcentrating or of separating the component to be separated, but solelyas a means of detection.

In summary, relatively small magnetic particles are not very appropriatefor separation, enrichment or concentration procedures, in traditionalprocedures, in particular immunoassays.

The document WO-A-96/09409 describes the use of the so-called MACS(Magnetic Cell Sorting) method, involving the formation of a complexbetween an analyte and a reagent comprising magnetic particles, forenriching/concentrating said analyte, in this instance fetalerythrocytes. These cells are then analyzed by flow cytometry, or usedas a source of genetic material, but without being immobilized on asupport, through bonding between the analyte and a receptor for example.

The document DE-A-4,036,288 describes a method which makes it possibleto detect and quantify one or more analytes specifically bound toparticles which are capable of being distinguished.

The subject of the present invention is a method, which makes itpossible to isolate an analyte from a liquid medium in which it isextremely dilute or not concentrated to any great extent. Moreparticularly, as regards the determination of the analyte, the subjectof the present invention is a method allowing a high sensitivity ofdetection and/or of quantification.

In accordance with the present invention, in general:

the reagent is brought into contact with a liquid sample obtained fromthe medium containing the analyte, to form an intermediate mediumcomprising, still in a discrete form and distributed in said medium, acomplex between said support, the receptor and the analyte,

the accessible surface of said other support is brought into contactwith at least one capture partner,

The deposit of the capture partner is concentrated by obtaining, fromthe intermediate medium, another sample, enriched with capture partner;and said other sample is brought into contact with the accessiblesurface.

A liquid stream, containing the capture partner, of limited section andadapted to the accessible surface, is established and said stream isbrought into contact with said accessible surface.

A liquid stream, containing the capture partner, is established and saidstream is brought into contact with said accessible surface, and thenrecycled in contact with the latter.

Before the description of the invention, it is appropriate to give adefinition of the terms used.

“Ligand” is understood to mean a component capable of forming, through achemical or physical bond, a complex with an analyte.

By way of example of ligand, there may be mentioned antibodies, antibodyfragments, antigens, haptens, lectins, sugars, ribo- anddeoxyribonucleic acids, proteins, in particular A or G, hormones,hormone receptors, biotin, avidin or strepta-,idin and, in general, itbeing possible for natural or synthetic ligands, and modified ligandanalogues to enter into competition with the ligands.

“Receptor” is understood to mean any ligand as defined above,immobilized on a support by any means such as adsorption, covalentbonding, chelation, molecular recognition, and the like, and capable ofretaining an analyte, alone or conjugated with another ligand.

“Support” is understood to mean any type of support, polymeric,inorganic or metallic. By way of example of polymeric supports, theremay be mentioned plastic supports based on polystyrene,poly(meth)-acrylates, polybutadiene, polypropylene, and the like, aloneor in the form of copolymers. By way of example of inorganic supports,there may be mentioned silicon oxide, silicon, mica, glass, quartz, andthe like. By way of example of metallic supports, there may be mentionedgold, silver, titanium oxide, vanadium oxide, and the like.

The immobilization of the ligands on the support may be carried outeither by simple adsorption on the native or modified support, or via achemical or physical reaction which makes it possible to modify thesurface of the support, and thus to allow the binding of the receptor bycovalent bonds, or other traditional means well known to persons skilledin the art.

“Limited surface” is understood to mean any surface obtained by chemicalor physical means, designed to reduce a surface of defined dimensions,for example by cutting into smaller-sized components, by covering with a“mask” which limits the initial surface to the inner contours of themask, or by chemical means of spreading based on a smaller surface, asdescribed in Kumar A. et al. (Langmuir (1994), 10, 1498-1511). Thereduction in the surface of the useful or active zone is not limited insize. Methods derived from microtechnologies make it possible, forexample, to obtain limited surfaces of micro or nanoscopic sizes, aswell as to have available and/or to convey micro or nanovolumes ofliquids to said useful zone of limited surface.

In the description below, the term “conjugated” will be reserved for theentity formed by immobilization between the ligand and the support ofthe reagent. The term “complex” will be reserved for the entity formedbetween the reagent, hypothetically conjugated, and the analyte. Whetherit is the support which is part of the reagent, the reagent itself, andthe complex, they are in a discrete form, distributed or dispersed in aliquid medium, and consequently in the form of particles.

The terms “support” and “other support” indicate that they arecomponents which are substantially of the same nature or havingessentially the same function. The term “support” will be reserved forthe support belonging to the reagent, and the term “other support”, tothe support of which at least part of the accessible surface or windowcontains one or more useful zones of limited surface.

The terms “receptor” and “other receptor” will be reserved respectivelyfor the ligands immobilized respectively on said support and said othersupport.

“Particle” is understood to mean any particle of a polymeric, inorganicor metallic support, on which it is possible to graft a ligand. Inparticular, the particles which may be separated by the action of anexternal physical means, for example by a magnetic or electrical route,or under the effect of gravity or by centrifugation are considered asfalling within the scope of the present invention. Falling outside thepreceding definition are the small-sized, in particularsuperparamagnetic, particles whose speed of sedimentation under theeffect of gravity is less than the thermal agitation, but which arecapable of constituting aggregates by any method which makes it possibleto combine them with each other, or to assemble them on larger-sizedparticles, which can be separated by any physical means.

By way of example of polymeric particles, there may be mentioned theparticles obtained by polymerization in emulsion such as latexes orlarger-sized particles, magnetic or otherwise.

By way of example of metallic particles, there may be mentionedcolloidal gold, ferro-, ferri-, para-or superparamagnetic particles,coated or otherwise with natural or synthetic polymers, whosecomposition comprises iron or other metals such as cobalt, nickel, aloneor in the form of alloys, magnetic or otherwise.

By way of example of inorganic particles, there may be mentionedparticles based on silica or silicon, magnetic or otherwise.

By way of example of methods of separation by an external physicalmeans, there may be mentioned sedimentation by gravity or bycentrifugation, magnetic attraction by the action of permanent magnets,of electromagnets, or by the use of devices which make it possible toincrease the magnetic field gradient, the electrical attraction, and anyother equivalent technique.

“Determination” is understood to mean any method which makes it possibleto detect the presence and/or to quantify the deposit concentrated inthe useful zone of the accessible surface of said other support, namelythe particles of the specific product of interest, that is to say of thecomplex or the reagent which has not reacted.

By way of example of a method of determination, there may be mentioned:

topographical methods such as atomic force microscopy, profilometry, andthe like,

magnetic methods as described, for example, in the documentFR-A-2,710,410, magnetic force microscopy, and any methods ofdetermination which are sensitive to the presence of ferro, ferri, paraand super paramagnetic metallic components

electrical methods such as the measurement of a variation incapacitance, described for example in the document EP-A-90 402 611,tunnelling microscopy, impedance measurements, and the like

optical methods which allow the detection, for example, of amodification of the thickness and/or of the refractive index (opticalthin layers, ellipsometry, surface plasmon resonance, surface acousticwaves, and the like) or the measurement of a light intensity (evanescentwaves, dark field microscopy, optical near field microscopy, and thelike)

methods which allow the measurement of mass variations (quartz crystalmicrobalance, and the like)

and in general, any techniques not cited here, but which are equivalent.

The present invention can accomodate various embodiments, depending onthe operating choices defined below.

In the concentration step, the capture partner concentration level isdetermined so that the quantity of said immobilized capture partnerremains at most equal to the quantity of said other receptor on saidother support. To this end, for example, the reagent comprises particlesof the support conjugated with the receptor, having the capacity to beseparated from any liquid medium in which they are dispersed, under theaction of a physical means applied to the liquid medium, and thisphysical means is applied to at least part of the intermediate medium,in order to separate the complex particles, these complex particles thusseparated are optionally washed, and the complex particles arerecovered, by ending the application of the physical means, in order tosubsequently obtain, in said useful zone, a deposit concentrated inrelation to the capture partner. By way of example, the particles of thesupport are magnetic, in particular superparamagnetic, and the physicalmeans consists of a magnetic field applied to the intermediate medium,for example according to the so-called HGMS (High Gradient MagneticSeparation) technique.

The receptor, that is to say the one belonging to the reagent, and saidother receptor, that is to say the one for the accessible capturesurface, contain the same ligand, immobilized respectively on thesupport and said other support.

When the reagent comprises particles of the support conjugated with thereceptor, having the capacity to be separated from any liquid medium inwhich they are dispersed, under the action of a physical means appliedto this liquid medium, this physical means is preferably applied inrelationship with the other support, that is to say the one for capture,in order to concentrate said deposit in any useful zone.

In some cases, the reagent is labeled, and the treatment of the analytecomprises a step of determining the marker for the deposit concentrated,immobilized in the useful zone of the other support, that is to say ofthe capture support. Conventionally, this labeling of the reagent isobtained according to one of the following modes, namely:

the support itself constitutes a marker; it is for example colloidalgold

the marker is bound to the support

the marker is bound to the receptor.

The analyte is determined from the deposit concentrated in the usefulzone, by any method chosen from the group including topographicalmethods, for example by atomic force microscopy (AFM), magnetic methods,for example by magnetic force microscopy, electrical methods, forexample measurement of a variation in capacitance, optical methods, forexample measurement of the refractive index, and finally methods formeasuring mass variation, for example quartz crystal microbalance.

More particularly, the useful zone is designed so that the quantity ofsaid deposit immobilized on said other support is at least equal to thesensitivity threshold of the method of determination, expressed asnumber of particles per unit of surface.

The assay format used is either direct, in which case said capturepartner is said analyte, or by competition, in which case said capturepartner is said reagent.

Preferably, another sample is obtained, by enriching said intermediatemedium with complex, and said other sample is brought into contact withthe accessible surface of said other support.

Various modes of bringing the other sample into contact with theaccessible capture surface are considered:

a measured quantity of said other sample is deposited on the accessiblesurface of the latter

or a stream of said other sample, of a relatively small thickness,optionally recycled, is caused to pass in contact with this accessiblesurface.

Finally, the liquid medium may contain a plurality of various analytes,and in this case the accessible capture surface of the other supportcomprises a plurality of useful zones, exhibiting a plurality of otherreceptors which are respectively different, of capture partners whichare respectively different.

The method according to the invention may be applied to a specificnucleic material of any bacterium, in this instance ribosomal RNA, inwhich case the sensitivity level obtained makes it possible to avoid anyenzymatic amplification method, such as PCR (Polymerase Chain Reaction),applied to the genomic material of the same bacterium, in order todetect the latter. Indeed, the sensitivity level obtained becomescompatible with the quantity of ribosomal RNA present in the bacterium,of the order of 10⁴ to 10⁵ molecules, which makes it possible todetermine the presence of this bacterium, without using amplification ofall or part of its genome.

DESCRIPTION OF THE FIGURES

FIG. 1 represents a general diagram of the formation of the complex insolution.

FIG. 2 shows an example of a device for separating the complex.

FIG. 3 represents a biospecific, accessible surface of the capturesupport. FIG. 4 represents the deposition of the solution containing thecomplex on the biospecific surface. FIG. 5 represents the reactionbetween the complex and the other receptors of the biospecific surface.

FIG. 4 represents an example of deposition of the solution containingthe complexes.

FIG. 5 represents an example of the reaction between the complex and thereceptor on the support.

FIGS. 6A and 6B schematically present two methods which make it possibleto assemble and immobilize particles of the complex on the biospecificsurface, one without concentration of the complex (FIG. 6A), and theother with concentration of the complex (FIG. 6B).

FIG. 7 represents two curves derived from the methods presented in FIGS.6A and 6B, respectively. The number of particles detected per unit ofsurface is represented on the y-axis, and the analyte concentration ofthe original liquid medium on the x-axis. The dotted line establishesthe sensitivity threshold.

FIG. 8 shows a surface accessible to the complex, belonging to saidother support, termed capture support, limited by the use of a mask.

FIGS. 9A and 9B present the deposition of particles of the complex onthe limited surface represented in FIG. 8, respectively without and witha means of concentrating, consisting of a magnet or electromagnet.

FIG. 10 represents a system identical to that represented in FIG. 4,with the limited surface represented in FIG. 8 and the addition of amagnet.

FIG. 11 represents the effect of enriching with complex by a magneticfield gradient, on the variation in the number of particles per unit ofsurface (μm²) on the y-axis, as a function of the TSH (analyte)concentration on the x-axis [Log10 ng/ml]. The black circles refer to anabsence of enrichment with a constant volume of 0.07 ml, the blacksquares to a variable volume concentrated by HGMS.

FIG. 12 shows four atomic force microscopy images which represent theeffect of the addition of a magnet during the reaction for the formationof conjugates between complexes and receptors of the biospecific oraccessible surface of the capture support. The figures on the left areobtained with concentration by a magnet, and the figures on the rightwithout concentration.

FIG. 13 shows the variation in the number of immobilized particles ofthe complex per unit of surface (μm²), as a function of the size of thebiospecific surface in mm² (on the x-axis).

FIG. 14A shows the variation in the number of immobilized particles ofthe complex per unit of surface (μm²), as a function of the original TSHconcentration in solution in pg/ml on the x-axis, for a small-sizedbiospecific surface; the figure on the right (FIG. 14B) shows theoriginal slope.

FIG. 15 compares the variation in the number of immobilized particles ofthe complex per unit of surface (μm²), as a function of the original TSHconcentration in solution, in ng/ml on the x-axis, according to the sizeof the biospecific surface. The black circles refer to a complex, firstenriched by magnetic field gradient HGMS, immobilized withoutconcentration on a biospecific surface of 64 mm².The black crosses referto an enriched complex, immobilized with concentration on a biospecificsurface of 0.5 mm². The concentration on the x-axis is in loglo (ng/ml).

FIG. 16 represents a comparison of the various modes of concentrationusing magnetic particles for the starting reagent. The y-axis and x-axisare the same, with the same scales, as those represented in FIG. 15. Theblack circles refer to a complex immobilized on the biospecific surface,without prior enrichment, or concentration at the time of capture on thebiospecific surface, the black diamonds to a complex immobilized afterenrichment on an HGMS columnn, and the black crosses to a compleximmobilized, after enrichment and with concentration.

FIG. 17 shows a comparison of the various modes of enrichment, usingcolloidal gold particles as a support belonging to the reagent. Thenumrber of particles per μm² is represented on the y-axis, the THSconcentration (Log 10 ng/ml) on the x-axis. The black diamonds refer toa determination after enrichment by centrifugation, and the blackcircles to a determination without enrichment by centrifugation.

FIG. 18 represents an embodiment of the invention for the assay ofhaptens by competition and FIG. 19 the expected theoretical curve,expressing, on the y-axis, the number of particles detected per unit ofbiospecific surface, and, on the x-axis, the original haptenconcentration.

FIG. 20 represents an accessible biospecific surface of the capturesupport, containing several useful zones with respectively differentaffinity receptors.

FIG. 21 represents another embodiment of the invention.

FIG. 22 represents another embodiment of the invention.

FIGS. 23 and 24 represent the embodiment described in FIG. 22 to whichthere have been added two means of concentrating, and a biospecificsurface containing several useful zones as described in FIG. 20, forexample.

FIGS. 25 and 26 represent another embodiment of the invention. Theypresent in particular the addition, to the surface of the magnet, of apointed parted part made of a so-called “soft” magnetic material havingthe propert of confining the magnet field line to the end of this tip.This part or the equivalent can equally well be added to the examplesdescribed in FIGS. 9B, 10, 18, 21, 23 and 24 or any other equivalentassembly.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As previously mentioned, a particular feature of the invention is amethod which makes it possible to isolate analytes, even when highlydiluted, from liquid media. The mode of isolation may be variable, anexample is given as a guide in order to facilitate understanding. Thismode has the advantage of using small-sized magnetic particles. FIG. 1shows an example thereof.

In a specific embodiment, the particles 1 used have a diameter ofbetween 10 and 1000 nm, preferably between 10 and 200 nm. They compriseat least one monocrystalline iron oxide core, having a diameter ofbetween 7 and 15 nm, itself surrounded by a layer of a hydrophilicpolymer (support), so as to avoid contact between the metallic core andthe ligand forming the receptor. The metallic core, given its size, hasa superparamagnetic character. Such suspensions are stable for severalmonths, without notable sedimentation. The polymer may be advantageouslyused for the immobilization or grafting of the ligands according tomethods known to persons skilled in the art. The polymer used here is apolysaccharide called Dextran®, having a molar mass of between 10,000and 2,000,000, preferably between 40,000 and 70,000. It may be activatedor functionalized, as appropriate, by various methods such as oxidationwith periodate, giving aldehyde functions by cleavage between vicinalalcohol functions, or with cyanogen bromide, and in general, by anyequivalent method.

The ligands 2 are either antibodies, antigens, oligonucleotides or anyother biological entity having the property of binding to an analyte, aswas defined above. The ligands may, for example, be coupled to thesurface of the particles, to the functions of the previously activatedpolymer, via primary amine functions existing in the native molecule orintentionally added to the ligand. The particle/ligand conjugate 3 isrepresented in FIG. 1.

FIG. 1 represents a general diagram of the formation of the complexes insolution. The conjugate 3 (reagent) is introduced into the mediumcontaining the analyte 4 to be separated. After an incubation timenecessary for the formation of the complex 5 between the conjugate andthe analyte, the solution (intermediate medium) is passed over theassembly represented in FIG. 2. This is an example of an enrichmentdevice based on the principle of a separation of components whichrespond to the influence of a magnetic field. It contains a separatingchamber 6 housing a matrix of filaments made of ferromagnetic material,such as steel filaments 7. This matrix maybe coated, as appropriate,with a polymer, as described in the documents U.S. Pat. No. 4,375,407 orWO-A-90/07380. The solution containing the analyte/reagent complexes 5is passed over the filamentous matrix placed in the magnetic field ofone or more magnets 8, at a flow rate which is adjustable and whichallows the complex to be retained by the matrix. Such a device makes itpossible to enrich the complex diluted in very large initial volumes.

After elimination of the foreign components and of the carrier liquid,the magnetic field is removed, and the concentrated complex is elutedwith a small volume of liquid. This volume is deposited on abiospecific, accessible surface represented in FIG. 3. It consists of aflat support 9 on which other receptors 10 specific for the analyte 4are immobilized.

A flat support such as silicon oxide has known advantages. There may bementioned, for example, a low roughness, adjustable sizes or variedmodes of functionalization. For example, the attachment of alkoxy or ofchlorosilanes allows the introduction of functions at the surface, withthe aim of immobilizing receptors in various ways (adsorption, covalentbonding, and the like), and the like. Other types of supports asdescribed above can however be used.

FIG. 4 represents an example of deposition of the solution containingthe complexes 5. It is eluted by removing the magnetic field and iseluted and deposited on the biospecific surface in a small volume.

FIG. 5 represents an example of the reaction between the complex 5 andthe receptor 10 on the support 9.

FIGS. 6A and 6B present one of the advantages of the invention. In FIG.6A, a given number of particles is concentrated on a so-called widebiospecific surface. FIG. 6B presents an improvement, in that the samenumber of particles is concentrated on a biospecific surface which issmaller in size than on the assembly in FIG. 6A.

FIG. 7 represents a theoretical curve showing the increase in the numberof particles per unit of surface, according to the assembliesrepresented in FIGS. 6A and 6B. If a sensitivity limit (dotted lines) isrepresented, it is advantageous to use a reduced surface, in that thisallows an improvement in the sensitivity threshold and therefore thedetection of lower concentrations of particles solution.

FIG. 8 shows an exemplary embodiment intended to limit the biospecificsurface constituted by a single useful capture zone. It is identical tothe biospecific surface represented in FIG. 4, but its size has beenlimited by the use of a mask 11 of controlled size.

In accordance with the preceding description, FIGS. 9A and 9B present aspecific carrying out of the deposition of particles on the reducedsurface represented in FIG. 8. FIG. 9B presents another advantage of theinvention. It is an improvement of the mode represented in FIG. 9A, bythe addition of a magnet 12 placed under the support and intended toconcentrate the complexes 5 on the usefuli zone 10.

FIG. 10 represents a system identical to that represented in FIG. 4,with the two refinements of the invention described above: the reductionin the accessible surface by the addition of a mask 11, and theconcentration or confinement of the particles on a surface of reducedsize by the addition of a magnet 12.

FIG. 25 and represent another embodiment of the invention. They presentin particular, in the lower part 20, the addition, to the surface of themagnet, of a pointed part of a so-called “soft” magnetic material havingthe property of driving and confining the magnet field lines to the endof this tip. This part or the equivalent may equally well be added tothe embodiments according to the examples described in FIGS. 9B, 10, 18,21, 23 and 24, or any other equivalent assembly. The upper part 19 isprovided with an inlet and with an outlet allowing the analytes 4 or thecomplexes 5 in the sample to be brought into contact with the receptors10 of the capture surface 13.

Additional details relating to the carrying out of the invention areindicated in the following examples.

EXAMPLES Example 1 Manufacture of Biospecific, Limited Surfaces byCoupling Anti-TSE (Thyroid Stimulating Hormone, or Analyte) Antibodies

The support used is composed of 8×8 mm silicon plates, coated withthermal silica. Possible organic contaminants are first removed bycleaning in the sulfochromic mixture. The plates are then immersed in 1ml of a 2% aminopropyldimethylethoxysilane solution (v/v) in toluene.The amine functions thus created on the surface are activated with 1 mlof disuccinimidyl suberate solution (10 mM in dimethyl sulfoxide).

The capture anti-TSH antibodies are diluted to 40 μg/ml in 50 mMphosphate buffer containing 0.15 M NaCl, pH 7.4 (PBS). 70 μl aredeposited on the activated plates. The coupling takes place for 1 hourat room temperature. The plates are then incubated for 2 hours at 37° C.with 70 μl of a solution of albumin at 0.1 mg/ml in PBS buffercontaining 0.5% Tween 20® (PBS/Tween), so as to avoid the adsorption ofundesirable molecules.

Example 2 Synthesis of the Magnetic Particles (free support)

This synthesis is described in the document U.S. Pat. No. 4,452,773(Molday), except for a few modifications.

14 grams of polysaccharide Dextran® T40 (Mw=40,000, Pharmacia) are addedto 14 ml of water, and the Dextran is left to dissolve at roomtemperature (solution 1). A solution 2 is prepared with 3 grams ofFeCl₃.6H₂O (Mw=270.3) and 1.3 grams of FeCl₂.4H₂ O (Mw=198.81) in 20 mlof water. Both solutions are introduced into a 250-ml jacketed reactorequipped with a stirring motor set at 200-250 revolutions/min, a glassstirrer and a dropping funnel containing a 7.5% NH₄OH solution (v/v). Atroom temperature, the NH₄OH solution is added dropwise, with stirring,until a final pH of between 10 and 11 has been obtained. The temperatureis brought to 70° C. for about 60 minutes, and then the final solutionis extensively dialysed against 5 liters of distilled water, withrenewal of the dialysis baths until a neutral pH has been obtained. Thesolution is then filtered on quartz wool in order to remove the largestaggregates, and then centrifuged 3 times at 600 revolutions/min for 5minutes. In order to remove excess Dextran, the particles are depositedon a (33×2.5) cm Sephacryl S300 HR gel column (Pharmacia), previouslyequilibrated in 0.1 M acetate buffer containing 0.15 m NaCl, 0.05% NaH₃,pH=6.5. The superparamagnetic particles obtained, coated with Dextran,have an outer diameter of between 20 and 900 nm, and preferably ofbetween 30 and 100 nm. They are stored at +4° C.

The assay of the particles is carried out by measuring the opticaldensity at 483 nm. A calibration straight line iron(II) salt of knownmolarity. The percentage by weight of iron in the particles is about 50(±10) %.

The assay of the Dextran is carried out by the technique described byMolday, R.S. et al (FEBS Lett., 170/2:232, 1973). The optical density ofthe complexes formed between the polysaccharide and the phenolsulfuricacid mixture is measured at 487 nm. The calibration curve is plotted onfree Dextran. The percentage by weight of Dextran in the particles isabout 50 (±10) %.

Example 3 Manufacture of Particle/Anti-TSH Antibody Conjugates, in Orderto Obtain the Reagent

3.1) Direct coupling of the anti-TSH antibodies to the particles ofExample 2

The particles are dialyzed in acetate buffer (0.1 M CH₃COONa, 0.15 MNaCl, pH=6.5). 5 μl/mg of particles in a 0.1 M sodium periodate solutionin the acetate buffer are added, and the reaction time is 45 minutes,protected from light. The particles thus oxidized are dialyzed in thecoupling buffer (0.25 M phosphate, 0.75 M NaCl), and the anti-TSHantibodies are added (10 to 100 μg/mg of particles). The coupling takesplace overnight at 37° C. with stirring.

The imine bond is then reduced by addition of sodium borohydride (0.1 M,1 μmol/mg of particles) for 15 to 30 minutes at room temperature, withstirring. The mixture is finally dialyzed against a 0.1 M phosphatebuffer containing 0.15 M NaCl at pH 7.4.

3.2) Use of commercially available particles coupled to streptavidin

3.2.1) Biotinylation of the anti-TSH antibodies:

2 mg of anti-TSH antibodies are dialyzed in PBS buffer, and then broughtinto contact for 1 hour with biotin-NSH (Pierce), with stirring, at roomtemperature (20 mol of biotin/mol of antibody). The biotinylatedantibodies are again dialyzed in order to remove the excess of biotin.

Example 4 Bringing of the Reagent into Contact with the Analyt, andEnrichment of the Intermeiate Medium Obtained

1.6×10¹⁰ particles of conjugates as obtained in Example 3 are diluted inPBS/Tween® in variable volumes (from 0.1 to 100 ml). 6 ng of TSH areadded to each solution and the assays are incubated f or 2 hours at 37°C., with stirring. Each solution is then deposited and enriched on thecolumn described in Example 3, and then washed with 1 ml of PBS/Tween inorder to remove the free antigens. To recover the particles, the columnis removed from the magnet. 30 μl of PBS/Tween are deposited andremoved, because their concentration in terms of particles is too low.70 μl are then deposited and collected.

Each sample of 70 μl is then deposited on a plate as obtained inExample 1. The solution is left to incubate for 1 hour at roomtemperature. The plates are then vigorously washed with PBS/Tween, andthen with water.

By way of comparison, assays are carried out with solutions of constantvolume (70 μl) in which there are mixed, as before, 1.6×10¹⁰ particlesof conjugate (reagent) and variable masses of TSH so as to obtain arange of concentrations comparable to those described above (from 0.06to 86 ng/ml). The solutions are not enriched on a column according tothe HGMS technique, but directly deposited on the plates as obtained inExample 1, and incubated under the same conditions as those describedabove.

The detection of the concentrated deposit of the superparamagneticcomplex particles, after specific reaction on the support, is carriedout by atomic force microscopy or AFM (Autoprobe, Park ScientificInstrument). The samples are dried with nitrogen, and analyzed in Two ofthe four plates are placed on a magnet consisting of rare earths(diameter 6 mm, height 25 mm, B=0.32 Tesla), the other two are outsidethe action of any magnetic field. 70 μl of solutions containing a TSHconcentration of 1 or of 100 ng/ml and an identical concentration ofconjugated particles (2×10⁹) are deposited on these plates. Thesolutions are left to incubate for 1 hour at room temperature, withstirring, so as to limit the nonspecific adsorption of the conjugatedparticles. The plates are then analyzed by AFM according to theprinciple described in Example 4, and the results are illustrated by theimages in FIG. 12. Although, in this specific embodiment, small-sizedmagnetic particles are used, the small volumes used allow, all the same,their separation by a permanent magnet because the separation distancesare small.

The two plates placed on the magnets show a density of particles whichis markedly greater than those for the plates not subjected to theinfluence of the magnetic field. It was demonstrated that the backgroundnoise (absence of TSH in solution) is comparable in both cases, andtherefore that the improvement in the antibody/antigen reaction on thebiospecific surface is attributable to the presence of the magnet. Theresults are presented in Table 1.

TABLE 1 Number of Number of TSH concentration particles/μm²particles/μm² (ng/ml) (without magnet) (with magnet) 1 0.3 8.0 100 33128

Example 6 Enrichment of the Intermediate Medium on an HGMS Column, andthen Application of a Magnet in Relationship With the BiospecificLimited Surface: Influence of the Size of the Biospecific Surface

The capture antibodies or ligands are coupled with the activated silicaon surfaces of increasing size. For this purpose, variable volumes of 1,10 and 70 μl of antibody solution are deposited at the center of theplates and cover surfaces of 1.8, 13 and 56 mm², respectively. Thesmallest surface (0.5 mm²) is made using a punched screen, placed at thecenter of the plate. The solutions of conjugated particles (7.6×10⁹) andof TSH (60 pg/ml) are incubated in 1 ml, and then purified on an HGMScolumn according to the protocol described in Example 4. The plates asobtained in Example 1 are then placed on the magnets, and the purifiedsolutions are deposited and left to incubate. Analysis by AFM allows thequantification of the number of particles per μm².

The results are described in FIG. 13. The number of complex particlesimmobilized per unit of surface increases when the biospecific surfacedecreases, and therefore when the complex particles are concentrated atthe center of the surface under the influence of the magnet. The smallerthe surface on which the complex particles can react, the greater theconcentration effect.

Example 7 Enrichment of the Intermediate Medium on an HGNS Column, andthen Application of a Magnet in Relationship With the Biospecific,Limited Useful Surface: Influence of the Quantity of Antigen (analyte)in Solution, and Evaluation of the Sensitivity Limit of the Method

The capture antibodies or ligands are coupled with activated silica on asurface of 64 or 0.5 mm². The conjugated particles (8×10⁹ per assay) andthe antigens (analyte) in variable concentration (from 0 to 500 nglml)are incubated in 1 ml, and then the complex particles are enriched on anHGMS column. The functionalized plates, that is to say coupled to theirown receptors, are then placed on magnets as shown in FIGS. 9B and 10,and the purified solutions are incubated, and then analyzed by AFM.

FIG. 15 makes it possible to clearly demonstrate once again theinfluence of the size of the biospecific useful surface, on the effectof concentration or confinement by magnetization. For the same TSHconcentration, a much higher density of complex particles is indeedobserved on the small surface (0.5 mm²), represented by crosses,relative to the large surface (64 mm²), for which the density isrepresented by circles.

For the concentration on a useful surface of 0.5 mm², the results aredescribed in FIG. 14. Calculation demonstrates that the value of theplateau obtained is in conformity with the expected theoretical curve(FIG. 7, curve 6B). The limit of detection is calculated as above(Example 6). according to the statistical method generally accepted: 10blanks (without antigen) are prepared, whose mean roughness (M_(R)) andstandard deviation (SD_(R)) are calculated. The limit of detection (LOD)is defined as being: LOD=M_(R)+3. SD_(R). This value is plotted on theslope at the origin of the calibration curve. A limit of detection of5×10⁻³ pg/ml, that is to say 1.2×10⁵ molecules of TSH is calculated(Table 2).

Comparison of the results:

FIG. 16 presents, on the same graph, the results described in Examples 4and 7 and makes it possible to demonstrate the improvement insensitivity provided by the assembly represented in FIG. 10. Table 2below also makes it possible to show the improvement in the sensitivitylimit for the detection of TSH, provided by magnetic enrichment on acolumn by field gradient on the one hand, and by the joint effect of themagnetic enrichment of the intermediate medium on a column by fieldgradient and of the magnetic concentration of the complex particles on asmall-sized biospecific surface, on the other.

TABLE 2 Limit of Useful detection surface Method (pg/ml) (mm²) Columnseparation by HGMS 100 64 HGMS column separation and 4 64 concentrationby magnetization HGMS column separation and 0.005 0.5 concentration bymagnetization on a small-sized surface

By way of comparison, the limit of detection obtained on automatedmachines for immunoanalysis is of the order of a few pg/ml depending onthe cases. A gain in sensitivity by a factor of about 100 to 1000 istherefore obtained using the method according to the present invention.

Example 8 Synthesis of the Conjugated Particles of Gold-anti-TSHAntibody

The example below shows that the method according to the invention canbe used and extended to particles of support which are nonmagnetic andof a different nature.

Solution of colloidal gold. The pH of the solution of colloidal gold(diameter of the particles: 15 nm, Polyscience, OD₅₂₀=0.524), isadjusted to 7.2 with a 0.2 M K₂CO₃ solution. The colloidal gold is thenfiltered on a 0.1 μm membrane.

Solution of ligand. The anti-TSH antibodies are dialyzed in 2 mM boratebuffer pH 9.00, and then filtered on a 0.1 μm membrane, just before use,in order to remove the possible aggregates, and assayed at 280 nm.

Synthesis of the protein/gold conjugated particles. The antibodysolution is added to the gold solution, with stirring, at aconcentration of 8 μg of antibody/ml of colloidal gold. After incubatingfor 10 minutes, colloidal stabilization is ensured by addition of abovine serum-albumin (BSA) solution at 10 mg/ml in PBS, in order toobtain a final concentration of conjugated particles of 100 μg/ml. Thesolution is filtered (0.1 μm), and then purified on a membrane with acut-off MWCO=300,000, in order to remove the excess of antibodies. Theconjugated particles are taken up in PBS/BSA/Tween 20 0.5% medium, andfiltered. They are stored at 4° C. in the presence of sodium azide(0.05%) and can be stored for several months. The sodium azide may beoptionally replaced by a sterilizing filtration at the time of use.

Example 9 Sandwich Assay of the TSH by the Conjugated Particles LabeledWith Colloidal Gold: Comparison of the Methods With and WithoutEnrichment by Centrifugation of the Intermediate Medium

Without enrichment

On plates as obtained in Example 1, 70 μl of TSH solution of increasingconcentration from 0 to 50 ng/ml of PBS/Tween 0.5% are deposited on theplates and incubated for one hour at 37° C. After rinsing withPBS/Tween, 70 μl of solution of conjugated particles (OD_(520 nm)=1.4)are incubated for 1 h at 37° C. The plates are then washed, dried andanalyzed by AFM. The number of complex particles per unit of surface iscounted.

With enrichment by centrifugation

After synthesis of the conjugated particles (reagent) described above,100 μl of conjugated particles (OD_(520 nm)=1.4) and 6 ng of TSH areincubated in variable volumes from 0.1 to 10 ml of PBS/Tween 0.5%containing 0.1 mg/ml of BSA. The solutions obtained are incubatedovernight at 370° C., with stirring. Controls without antigen (analyte)are prepared. After the incubation, the solutions are enriched bywashing and membrane filtration (MWCO=100,000) in order to obtain afinal volume of a few tens of μl. They are then deposited on plates asobtained in Example 1. The incubation takes place for 1 h at 37° C. Therinsing and the analysis take place as above.

The results are presented in FIG. 17. A clear improvement in thesensitivity at equivalent TSH concentration is observed when the complexparticles are concentrated.

Example 10 Manufacture of the Biospecific Supports (other supports andreceptors) by Coupling of Oligonucleotides (ODN)

The silica surfaces are cleaned with sulfochromic acid (2 h, 120° C.)and then silanized with aminopropyldimethylethoxysilane (2% in anhydroustoluene) for 2 h at room temperature. A copolymer, maleicanhydride-co-methyl vinyl ether (MAVE, Mn=20,000), capable ofestablishing covalent bonds with amine functions, is then grafted on theamine-containing surfaces. A solution containing 1 mg of MAVE/10 ml ofanhydrous DMSO is prepared just before use. The amine-containingsurfaces are immersed in this solution and incubated, with stirring, for1 h at room temperature in the presence of triethylamine (1% v/v). Theyare then rinsed with DMSO and dried with nitrogen.

A solution of capture ODN (17 bases) is prepared at a concentration of 8μM in a 0.1 M sodium borate, 0.5 M NaCl (5%)/DMSO (95%) mixture. 50 μlof solution are deposited on the plate and incubated for 1 h at 37° C.The supports are then rinsed in PBS-Tween. Next, 50 μl of a solution ofPEG are deposited on the surface and incubated for 30 minutes in orderto avoid nonspecific interactions. The surfaces are rinsed withPBS/Tween. Such surfaces are called hereinafter “biospecific surfaces”.

Example 11 Manufacture of the Magnetic Particles/ODN Biotin DetectionConjugates Example 11a

100 μl of magnetic particles coated with streptavidin (Immunicon) areadded to 100 μl of a solution of the biotinylated detection ODN (21bases) (8.37 pmol/μl). This solution is incubated for 1 h at 37° C.,with stirring. The excess of ODN is removed by 3 successivemagnetization/dilution steps and the conjugates are finally taken up in100 μl of PEG buffer.

Example 11b

100 μl of magnetic particles coated with streptavidin (10¹¹ particles,that is to say 5×10¹² particular, in the lower part 20, the addition, tothe surface of the magnet, of a pointed part of a so-called “soft”magnetic material having the property of driving and confining themagnet field lines to the end of this tip. This part or the equivalentmay equally well be added to the embodiments according to the examplesmolecules of streptavidin, Miltenyi Biotec) are added to the solution ofthe biotinylated detection ODN (28 bases) (4×10¹³ molecules), carrying aspacer strand of eight bases. This solution is incubated for 1 h at 37°C., with stirring. The excess of ODN is removed by passing over an HGMSseparation column and then taken up in 100 μl.

The two reagents thus obtained are called hereinafter “detectionconjugates”

Example 12 Capture of DNA Targets (analyte) by the Detection Conjugatesof Example 11a

20 μl of the solution of detection conjugates as described in Example11a are diluted in PEG buffer. The targets (37 bases, 8.23×10¹³copies/μl), carrying the sequences respectively complementary to thecapture and detection ODN, are added in the quantity desired for a totalvolume of 1 ml. The mixture is incubated for 2 h at 37° C., withstirring. The complexes formed will be used as they are.

Example 13 Concentration of targets (analyte) With the Aid of theDetection Conjugates of Example 11a. Influence of the Presence of theMagnet

2 biospecific supports are prepared. Also, 2 samples, each containing10¹² copies/ml, are incubated in the presence of detection conjugates asdescribed in Example 11a. The biospecific supports are placed at thebottom of a vessel. One of these vessels is placed on a magnetconsisting of rare earths (diameter 6 mm, height 6 mm, B=0.32 Tesla).The solution containing the targets hybridized with the magneticparticles is deposited in the vessels, and incubated for 1 h, withstirring, by pipetting every 10 min. After the incubation, the supportsare rinsed in PBS-Tween and then in 0.1 M ammonium carbonate buffer,before being dried and analyzed in contact with air mode by AFM.

In a useful zone of 4×4 μm of the biospecific surface, 0.3 particle perμm² is detected without a magnet, and 86 particles per μm² with amagnet.

The targets are practically not detected in the absence of the magnet,whereas a monolayer of particles is obtained in the presence of themagnet, corresponding to the saturation of the signal.

Example 14 Concentration of Targets (analyte) With the Aid of DetectionConjugates of Example 11a . Determination of the Limit of Detection

Several biospecific supports are prepared. Increasing concentrations oftargets (between 0 and 8×10¹² targets/ml) are prepared in 1 ml of PEGbuffer. The capture of the DNA targets by the detection conjugates asdescribed in Example 11a is carried out as described in Example 12.After the incubation, the biospecific supports are placed on magnets asdescribed in Example 12, at an incubation temperature of 37° C. 100 μlof the solutions containing the targets hybridized with the detectionconjugates are deposited on the biospecific supports. The solution isincubated for 3 minutes and then the supernatant is removed. 100 μl areagain deposited, and then the drop is stirred by pipetting in order toresuspend the particles. This protocol is repeated until all thesolution has been concentrated on the support (10×100 μl). After theincubation, the surfaces are rinsed with PBS-Tween and then with 0.1 Mammonium carbonate buffer, before being dried and analyzed in contactwith air mode by AFM. The density of the particles is measured in themaximum magnetization zone. The level of covering of the particles onthe images is determined by the use of image processing software.

The sensitivity limit is estimated at 7×10⁸ targets/ml, that is to say again by a factor of 100 relative to the detection of DNA targets by aconventional enzymatic test, of the ELOSA type (Enzym Linked OligoSorbent Assay), without concentration (8×10¹⁰ targets/ml), carried outon the same support.

Example 15 Concentration of Targets (analyte) With the Aid of theDetection Conjugates of Example 11b. Dotermnation of the Limit ofDetection

50 μl of the solution of detection conjugates as described in Example11b are diluted in PEG buffer. The targets (37 bases), carrying thesequences respectively complementary to the capture and detection ODN,are added in the desired quantity for a total volume of 1 ml. Themixture is incubated overnight at 37° C., with stirring. The detectionconjugate-target complexes are then purified on an HGMS separationcolumn as described in Example 3, in order to obtain a final volume of100 μl.

Sever al biospecific supports are prepared. They are placed on magnetsas described in Example 13, at an incubation temperature of 37° C. Thesolution of detection conjugate-target complexes is incubated for 30minutes with stirring by pipetting every 10 minutes. After theincubation, the surfaces are rinsed with PBS-Tween and then 0.1 mammonium carbonate buffer, before being dried and analyzed in contactwith air mode by AFM.

The limit of detection is estimated at 3×10⁹ copies/ml, that is to say again by a factor of 30 with respect to the ELOSA test carried out on thesame support without concentration.

Example 16 Comparison of the Concentrating System in Solution, WithRespect to a Sequential Incubation of the Reagents at the Surface

Assay 1: the targets are captured in solution as described in Example15.

Assay 2: after preparation of the biospecific supports, 100 μl of asolution of targets (between 0 and 8×10¹³ targets/ml) are incubated onthe support (particles) which is then rinsed with PBS-Tween. Next, 100μl of solution of biotinylated ODNs (0.5 μM in PEG) are incubated.Finally, the support is placed on a cylindrical magnet as described inExample 13, and 100 μl of a commercially available solution of magneticparticles coated with streptavidin (Miltenyi Biotec) and dilutedone-half with the PEG buffer are incubated for 30 minutes with stirringevery 10 minutes.

The detection limit of the sequential system at the surface is estimatedat 7.6×10¹⁰ targets/ml, whereas that of the assay in solution wasestimated in Example 15 at 3×10⁹ copies/ml. The amplification factorlinked to the incubation of the targets and of the conjugates insolution is therefore 25.

Example 17 Advantage of the Flow-through Cell

With the Aim of Concentrating Magnetic Conjugates

This assay illustrates another method of carrying out the inventionrepresented in FIGS. 23 and 24.

Biospecific supports are prepared by adsorbing biotinylated (positivecontrol) or nonbiotinylated (negative control) antiferritin antibodiesat a concentration of 0.2 μl/ml on silica surfaces.

Assay 1: These supports are placed in the cell containing a cylindricalmagnet as defined above, surmounted by a truncated cone 0.5 mm indiameter at its summit. A solution containing 30 μl of magneticparticles coated with streptavidin (Miltenyi Biotec), diluted in 1 ml ofPBS/Tween/BSA buffer (0.1 mg/ml), is injected onto each support at aflow rate of 0.017 ml/min. After this incubation, the supports arerinsed, dried and analyzed by AFM.

Assay 2: In parallel, assays are carried out on these same biologicalsupports outside the cell. Each support is placed on a cylindricalmagnet as defined above. A solution containing 30 μl of magneticparticles coated with streptavidin (Miltenyi Biotec), diluted in 60 μlof PBS-Tween-BSA buffer is prepared and deposited on the supports, andthen incubated for 30 minutes by stirring every 10 minutes. After thisincubation, the supports are rinsed, dried and analyzed by AFM and thelevel of covering of the particles is evaluated.

It is observed i) that the specific signal (97+/−3 for Assay 1, 63+/−16)for Assay 2 and ii) and the signal/noise ratio (139 for assay 1, 31 forassay 2) are markedly improved during the use of the flow-through cell.

In summary, the examples described above embody the general methodaccording to the invention. Example 7 shows that the invention makes itpossible to detect analyte concentrations as low as 5 fg/ml of TSH,which corresponds to 1.2×10⁵ molecules. A bacterium represents about 10⁴to 10⁵ molecules of ribosomal RNA. Examples 10 to 16 show that methodsof implementation, which are identical or different, can be used for thedetection of DNA targets. Consequently, with an implementation of thesame type, it appears possible to detect from 1 to 10 bacteria per unitof volume. Associated with a method of concentration as described andapplied to nucleic material, the analytical sensitivity of the methodaccording to the invention is therefore of the same order as that ofmethods of amplification such as PCR, NASBA, and the like.

The embodiments of the invention may be many and, without beinglimiting, a few examples thereof may be given:

The invention may be applied to the assays of haptens by competitionaccording to an example of a method described in FIG. 18. Haptens areimmobilized on a limited surface. The solution containing haptens to beassayed (analyte) is placed in contact with conjugated particlescarrying only one anti-hapten antibody (receptor) as described inExample 3, for a sufficient time for the complex particles to form. Thecomplex particles are separated according to one of the methodsdescribed and concentrated on the useful surface carrying the receptorhaptens. The conjugated particles which have previously reacted with ahapten in solution cannot bind and are removed by washing. Theconjugated particles which have not reacted, carrying the availableantibody may, in contrast, react with the receptor haptens of thebiospecific useful surface and be detected by one of the various meansdescribed above. The number of conjugated particles detected decreasewhen the hapten concentration in solution increases, as is representedon the theoretical curve of FIG. 19.

As described in the documents U.S. Pat. No. 5,543,289 or U.S. Pat. No.5,169,754, cells may be separated from a solution by magnetic sorting.Following this operation, it is possible to concentrate the labeledanalyte (cells, yeasts, bacteria, viruses or phages) and to retain it ona small-sized biospecific surface, by a receptor different from oridentical to that which allowed the production of the complex particles,by a method as described in FIG. 10, for example. The determination(detection and/or quantification) may be made by any appropriate means,such as intrinsic or extrinsic fluorescent markers, microscopy methods,and the like, including the detection by the use of particles which haveserved for the separation and concentration.

Specific embodiments, intended in particular to bind several ligandshaving different affinities on the same limited surface (multiaffinity),and/or to concentrate the conjugated particles at desired locationsmakes it possible to apply the invention to a multiple detection. Anexample is described in FIG. 20; in this case, the useful surfacecomprises three useful zones A, B and C comprising three receptors whichare respectively different, and situated at three identified geographicpositions. Using a specific method of implementation, as described inFIG. 21, conjugated particles are immobilized on their specificreceptors. The method comprises a device 14 which comprises acompartment 15 in which the formation of the complex particles 5 takesplace. This compartment may comprise a system for mechanical stirring,or, if the particles used are magnetic, a magnetic field stirringsystem. After a sufficient time for the formation of the complexparticles, these are brought into contact with the surface 17 (mono ormultiaffinity) via a channel 16; the latter, of low height, is intendedto limit the height of the solution above said surface, thus limitingthe distance which a complex particle has to travel before beingcaptured by its specific receptor. The probability of a complexparticle/receptor reaction may be advantageously improved byrecirculating the solution on the limited surface. The device describesthe use of a magnet 12 which moves from one position to the next (forexample, from zone A to zone B, then to zone C). A single magnetoccupies, according to another variant, the entire surface, or severalmagnets, each facing a zone A, B or C, without this changing the methodof the invention. The device described may consequently be used for themultidetection of analytes, or for the screening of libraries (phagelibraries, for example).

The devices described above can be used according to techniques derivedfrom microtechnologies.

What is claimed is:
 1. A method for at least one of isolating, detectingand quantifying at least one analyte distributed in a dilute liquidmedium, comprising the following steps: a) providing (i) a first reagentcomprising particles of a first support distributed in the liquid mediumand at least one first receptor for an analyte fixed on said particles;and (ii) a capturing means comprising a second support having an exposedsurface defining at least one active zone and at least one secondreceptor for said analyte or for said first reagent, said secondreceptor being fixed in said active zone; b) contacting a liquid sampleobtained from the medium containing the analyte with said first reagentto obtain an intermediate reagent comprising particles of a complexdistributed in another liquid medium, said complex being the reactionproduct of at least part of said first reagent with said analyte; c)contacting the capturing means with at least one capture partnerselected from the group consisting of the part of the first reagent thatwas not reacted with said analyte during step (b) and the intermediatereagent and binding the capture partner to the active zone; and d)increasing the quantity of capture partners exposed per unit of surfaceof the active zone, wherein a concentrated deposit of the capturepartner is immobilized in the active zone, whereby small quantities ofanalyte in said dilute medium are isolated, detected or quantified. 2.The method according to claim 1, wherein the capture partnerconcentration level is determined so that the quantity of saidimmobilized capture partner remains at most equal to the quantity ofsaid second receptor on said second support.
 3. The method according toclaim 2, wherein the first reagent comprises particles of the firstsupport conjugated with the first receptor having the capacity to beseparated from any liquid medium in which they are dispersed, under theaction of a physical means applied to the liquid medium, said methodfurther comprising applying said physical means to at least part of theintermediate medium to separate said complex particles, optionallywashing said separated complex particles and eluting said complexparticles by ending the application of said physical means to increasethe ratio of concentration of the capture partner to said deposit insaid active zone.
 4. The method according to claim 3, wherein theparticles of the first support are magnetic or superparamagnetic and thephysical means comprise a magnetic field applied to the intermediatemedium.
 5. The method according to claim 3, wherein said particles arenon-magnetic and said physical means are selected from the groupconsisting of: electrical, gravity, and centrifugation.
 6. The methodaccording to claim 4, wherein said physical means applied to saidmagnetic particles is High Gradient Magnetic Separation (HGMS)technique.
 7. The method according to claim 1, wherein said firstreceptor and said second receptor contain the same ligand immobilizedrespectively on said first support and said second support.
 8. Themethod according to claim 1, wherein the first reagent comprisesparticles of the first support conjugated with the receptor having thecapacity to be separated from any liquid medium in which they aredispersed under the action of a physical means applied to said liquidmedium, said physical means applied in relationship with said secondsupport, to concentrate said deposit in said active zone.
 9. The methodaccording to claim 1, wherein the first reagent is labeled and thetreatment of the analyte comprises a step of determining the marker forthe deposit, which is concentrated and immobilized in said active zoneof said second support.
 10. The method according to claim 9, wherein themode of labeling of the first reagent is selected from the groupconsisting of: the support itself is a marker; a marker is bound to saidsupport; and a marker is bound to said receptor.
 11. The methodaccording to claim 9, wherein the analyte is determined from the depositconcentrated in said active zone by the method selected from the groupconsisting of topographical methods, magnetic methods, electricalmethods, optical methods and methods for measuring mass variations. 12.The method according to claim 11, wherein the active zone is designed sothat the quantity of the deposit immobilized on said second support isat least equal to the sensitivity threshold of the method ofdetermination, expressed as number of particles per unit of surface. 13.The method according to claim 11, wherein said topographical methodscomprises: atomic force microscopy (APM) or profilometry.
 14. The methodaccording to claim 11, wherein said electrical methods comprises:measurement of a variation in capacitance or resistance, tunnelingmicroscopy, or impedance measurements.
 15. The method according to claim11, wherein said optical methods comprise measurement of the refractiveindex or the measurement of light intensity.
 16. The method according toclaim 11, wherein methods for measuring mass variations comprise quartzcrystal microbalance.
 17. The method according to claim 1, wherein thecapture partner is said intermediate reagent and the amount of analytesis determined by a direct assay format.
 18. The method according toclaim 1, wherein the capture partner is said first reagent and theamount of analytes is determined by a competitive assay format.
 19. Themethod according to claim 1, wherein another sample is obtained byenriching said intermediate medium with complex and said other sample isbrought into contact with the accessible surface of said second support.20. The method according to claim 19, wherein said other sample isbrought into contact with the accessible surface of said second supportby depositing a measured quantity of said other sample on the accessiblesurface.
 21. The method according to claim 19, wherein said other sampleis brought into contact with said accessible surface of said secondsupport by passing a stream of said other sample, of a relatively smallthickness, optionally recycled, in contact with said accessible surface.22. The method according to claim 1, wherein the liquid medium containsa plurality of various analytes, wherein the accessible surface of saidsecond support comprises a plurality of active zones, having a pluralityof different receptors for different capture partners.